Free radical biochemistry is an important and emerging new area probing the involvement of organic free radicals, molecules having unpaired electrons, in biochemical processes. Historically, studies on free radicals in biology have emphasized the deleterious effects of radicals in toxicity mechanisms and radiation damage, but there is a growing recognition of the essential role that free radicals play in enzyme catalysis and the biological function of redox metalloproteins. Early studies in the area of free radical enzymology focussed on Fe- and B12- dependent ribonucleotide reductases and mutases from certain anaerobic bacteria. However, the significance of radicals in biochemical mechanisms is now well established in enzymes with as important and diverse functions as prostaglandin and penicillin biosynthesis, lipid peroxidation and alkane oxidation. A motif which is emerging from these studies is the association of the radical site with a metal center that is involved in generation, stabilization or control of reactivity of radicals in proteins. Two of the most well-defined free radical metalloenzymes are galactose oxidase and the Fe-dependent ribonucleotide reductase RRB2. Each of these enzymes stabilize a protein side chain as a free radical in the resting enzyme, a radical which is required for catalytic activity. Extensive biochemical and basic spectroscopic characterization is now complete for both proteins, and both are also crystallographically defined at near atomic resolution. Detailed spectroscopic studies of these proteins can now be expected to extend to geometric information available in the crystalligraphic data to develop the special electronic structural features of the radical sites and metal interactions, leading to insights into the mechanism of stabilization of radicals in proteins and the control of their unique reactivity. these key aspects of free radical biochemistry have not been previously defined by protein structural studies. These key aspects of free radical biochemistry have not been previously defined by protein structural studies. Our studies will focus on the information contained in electron paramagnetic resonance (EPR), electronic absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of the active site complexes in galactose oxidase (GO), ribonucleotide reductase (RRB2) and isopenicillin N synthase (IPNS). In the former (GO, RRB2) we will probe the radical sites as well as the biological metal complex. For IPNS the resting ferrous enzyme and its substrate complex will be of key interest in terms of radical-generating species. In all three studies, inorganic models will provide important calibration. To probe the mechanisms of reactivity for free radical metalloenzymes we will explore chemical perturbations, examining the spectroscopic and electrochemical consequences of coordinating exogenous ligands in the active complexes, and investigate the details of turnover reaction using transient kinetics methods. These fundamental studies will form the basis for characterization of other important and interesting free radical metalloenzymes.